Unmasking the phospholipid-induced “hook effect” and its implications - increasing phospholipid can also lead to a longer clotting time in coagulation assays

It is generally accepted that higher phospholipid concentrations generate shorter clotting times. Phospholipid-dependent lupus anticoagulants (LA) assays such as dilute Russell's viper venom time (dRVVT) and silica clotting time (SCT) have therefore been developed. However, cases have been observed where LA confirming tests (concentrated phospholipid) generates longer clotting times than screening tests (diluted phospholipid). This study investigates the underlying cause of this paradox and assess its implications. With different phospholipid concentrations, Russell's viper venom and/or silica-induced clotting times were assayed in normal pooled plasmas (NPPs), factor deficient plasmas, cirrhotic patients' plasmas (CPPs), LA positive plasmas (LPPs). Additionally, routine LA assays were performed in LPPs with or without factor deficiency. In NPPs and factor deficient plasmas, higher phospholipid concentrations resulted in shorter clotting times, however, this effect was more evident with low-middle phospholipid than with higher phospholipid (a U shape curve). In CPPs, clotting time was increasing along with increasing phospholipid from the beginning (right part of a U shape curve). In LPPs, clotting time was shortening along with increasing phospholipid at the beginning, but changeless thereafter (left part of a U shape curve). Compared to LPPs without factor deficiency, LPPs with factor deficiency demonstrated a smaller correction of screening by confirming (dRVVT, 25.9% versus 15.1%, SCT, 28.6% versus 4.1%), leading to a possibility of LA misdiagnosis. Increasing phospholipid could induce "hook effect" in coagulation assays, therefore, phospholipid-dependent clot-based coagulation assays such as LA assays need careful interpretation, especially among patients suffering coagulation factor deficiency.